During
testing
of
voluntary
ethanol
(EtOH)
consumption
one
25
mL
graduated
cylinders
always
contained
tap
water
and
the
other
contained
diluted
solutions
of
EtOH

3

0

7-9

-

3

To
control
for
side
bias,
the
positions
of
the
EtOH
cylinders
on
Days
1
and
2
are
switched

3

0

7-9

water
and
3%
EtOH
consumption

5-8

Graduated
cylinders
containing
EtOH
are
replaced
and
their
positions
on
Days
3
and
4
are
switched

6

0

8-9

water
and
3%
EtOH
consumption

7

The
positions
of
the
EtOH
within
a
group
on
Days
5
and
6
are
switched

6

0

8-9

water
and
6%
EtOH
consumption

8

Testing
cages
are
changed

6

0

8-9

water
and
6%
EtOH
consumption

9-12

Graduated
cylinders
containing
EtOH
are
replaced
and
their
positions
on
Days
7
and
8
are
switched

10

0

9-10

water
and
6%
EtOH
consumption

11

The
positions
of
the
EtOH
cylinders
on
Days
9
and
10
are
switched

10

0

9-10

water
and
10%
EtOH
consumption

13

Graduated
cylinders
containing
EtOH
are
replaced
by
0.2%
saccharin
solutions
and
their
positions
on
Days
11through
12
are
switched

0

0.2

9-10

water
and
0.2%
saccharin
intakes

14-17

Graduated
cylinders
containing
only
saccharin
are
replaced
with
solutions
containing
EtOH
and
saccharin

3

0.2

10-11

water
and
0.2%
saccharin
intakes

16

The
positions
of
the
EtOH
and
saccharin
on
Days
13
through
15
are
switched
and
cages
are
changed

3

0.2

10-11

water
and
3%
EtOH
plus
saccharin
consumption

18-21

Graduated
cylinders
containing
EtOH
and
saccharin
are
replaced
and
their
positions
on
Days
16
and
17
are
switched

6

0.2

11-12

water
and
3%
EtOH
plus
saccharin
consumption

20

The
positions
of
the
EtOH
and
saccharin
on
Days
18
through
19
are
switched

6

0.2

11-12

water
and
6%
EtOH
plus
saccharin
consumption

22-25

Graduated
cylinders
containing
EtOH
and
saccharin
are
replaced
and
their
positions
on
Days
20
and
21
are
switched

10

0.2

11-12

water
and
6%
EtOH
plus
saccharin
consumption

24

The
positions
of
the
EtOH
and
saccharin
on
Days
22
through
23
are
switched
and
cages
are
changed

10

0.2

11-12

water
and
10%
EtOH
plus
saccharin
consumption

Equipment

•
25
mL
graduated
cylinders
fitted
with
metal
sipper
tubes

Reagents,
supplies,
solutions

Ethanol
(EtOH)
(
3,
6,
and
10%
;
v/v
solutions
with
tap
water)

Saccharin
(0.2
%
solution
in
tap
water)

0.2%
saccharin
+
ethanol
(EtOH,
3,
6,
10%
solutions)

Procedures

a.
Mice
5-6
wks-of-age
are
acclimated
to
housing
conditions
for
3
wks.
Initially
they
are
grouped
housed
at
3-4
mice
per
cage.b.
Mice
are
separated
and
housed
individually
for
1
day
to
be
acclimated
to
testing
conditions.c.
During
the
entire
day
of
acclimation
the
mice
are
continuously
exposed
to
2
inverted
graduated
cylinders
with
metal
sippers
and
food.d.
EtOH
with/out
0.2%
saccharin
in
the
same
25
mL
graduated
cylinders
are
placed
in
two
empty
cages
for
measuring
evaporation
and
leakage.
The
average
volume
depleted
from
these
cylinders
is
subtracted
from
individual
daily
consumption.e.
Mice
are
first
exposed
to
increasing
concentration
of
EtOH,
then
0.2%
saccharin
in
tap
water,
and
then
combination
of
EtOH
and
0.2%
saccharin.f.
Each
novel
solution
is
given
for
4
days,
and
each
morning
daily
fluid
consumption
(to
the
nearest
0.2
mL)
is
recorded
from
the
tap
water
cylinder
and
the
test
cylinder.g.
The
cylinder
positions
are
alternated
every
other
day
to
control
for
side
bias
while
food
is
distributed
in
equal
proximity
to
the
cylinders
to
avoid
preference
associated
with
food.h.
With
each
solution
change,
fresh
tap
water
is
provided
and
body
weight
is
concurrently
obtained.i.
Soiled
cages
are
changed
every
8
days.

Data
collected
by
investigator

Average
fluid
consumption
on
the
2nd
and
4th
day
of
each
set
of
paired
solutions
per
given
condition
(3%,
6%,
10%
EtOH,
0.2%
saccharin,
EtOH
+
0.2%
saccharin).

Definitions
and
calculations

Two-bottle
choice
test:
A
component
of
choice
preference
test
wherein
mice
are
offered
two
graduated
cylinders-one
with
3%,
6%
or
10%
EtOH
(with/out
saccharin)
and
the
other
one
with
water.
Side
bias
is
taken
into
consideration
by
alternating
the
position
of
the
EtOH
relative
to
water
cylinders.